EDMAN SEQUENCING RESEARCH GROUP 2004 STUDY: MODIFIED AMINO ACIDS IN EDMAN SEQUENCING

Edman degradation sequencing relies on comparing high-performance liquid chromatography retention times of the sample phenylthiohydantoin amino acids with phenylthiohydantoin amino acid standards. The elution characteristics of the twenty common amino acids have been well characterized, which aids in making confident assignments. Modified amino acids may present more of a challenge since they are not part of the commonly used standards and because the protein sequencer analyst may not have experience with them. Laboratories requesting a sample were sent a tube containing approximately 775 pmoles of a 20-amino-acid synthetic peptide composed of several modified amino acids that may be found in proteins or are generated during sample preparation. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the Edman Sequencing Research Group website (ESRG). The goal of the study consisted of two parts: assessment of the ability to correctly assign all the amino acids in the peptide, including the modified amino acids; and the collection and compiling of elution time characteristics of modified amino acids for instruments used in the study. The resulting compilation of the modified amino acid elution times and running conditions will be accessible at the Association of Biomolecular Resource Facilities (ABRF) ESRG website for future reference. The ABRF ESRG 2004 sample is the 16th in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.